This invention relates to the cloning and isolation of the dog gonadotropin-releasing hormone (GnRH) receptor, and also to mutant or polymorphic forms of the receptor and recombinant nucleic acids encoding the same. The invention also relates to genetically engineered host cells which express the receptor, antibodies against the receptor and polypeptides thereof. The invention also relates to uses of the receptor, recombinant nucleic acids and recombinant host cells in drug screening and development, diagnosis and therapeutic applications.
Gonadotropin-releasing hormone (GnRH) plays a pivotal role in the control of reproduction. It is a neuronal decapeptide hormone released from hypothalamus in a pulsatile manner. GnRH interacts with its receptor on the gonadotropes in the anterior pituitary and which, in turn, activates phospholipase C (PLC) via a pertussis toxin-insensitive G protein, Gq/G11. Two second messengers, inositol trisphosphate (IP3) and diacylglycerol (DG), are formed from the hydrolysis of phosphoinositide bisphosphate by PLC. IP3 and DG then act either separately or in concert, via increase of intracellular Ca2+ and activation of protein kinase C, to regulate the synthesis and release of both luteinizing hormone (LH) and follicle stimulating hormone (FSH). LH released from the pituitary gland is primarily responsible for the regulation of gonadal steroid production in both sexes, whereas FSH regulates spermatogenesis in males and follicle development in females.
The GnRH receptor (GnRH-R) is mainly expressed in the pituitary gland. It was also detected in extrapituitary tissues such as brain, breast, gonads, and ovarian tumors. The GnRH receptor has been cloned and sequenced from several mammalian species including human, ovine, bovine, pig, rat, and mouse. The cloning and expression of the murine and human receptors has recently been described in U.S. Pat. No. 5,750,366. The GnRH receptor from bovine, cow, sheep, and human contains 328 amino acids, while the rodent receptor has 327 amino acids, due to a deletion of a residue in the second extracellular domain. Analysis of the primary sequence identifies the GnRH receptor as a member of the G protein-coupled receptor (GPCR) family with seven transmembrane (TM) domains. However, the mammalian GnRH receptors have several unique structural features compared with other GPCRs. These include (1) the lack of the entire intracellular C-terminal tail; (2) the replacement of Tyr by Ser in the conserved G protein signature DRY motif of the proximal second intracellular domain; and (3) the reciprocal exchange of two amino acids, Asp in TM II and Asn in TM VII, that are highly conserved in most other GPCRs. In addition to pituitary gland, the expression of GnRH receptor message has also been demonstrated in extrapituitary tissues such as brain, breast, gonads, and ovarian tumors. The receptor sequences obtained from extrapituitary sources were identical to the corresponding pituitary GnRH receptor cDNAs.
Synthetic peptidyl GnRH analogues have been widely used in treating patients with prostate and breast cancers, endometriosis, uterine fibroids, precocious puberty, and other endocrinological disorders. Continuous infusion of high dose GnRH agonists internalizes the GnRH receptor and subsequently desensitizes the receptor, therefore effectively reducing the levels of LH and FSH. However, GnRH agonists can cause flare of the disease at the initial use due to their stimulatory effects. Cetrorelix and other peptide antagonists which are under development have been shown to reduce the levels of LH and FSH without the flare effect. Non-peptidyl GnRH antagonists have been disclosed in U.S. Pat. Nos. 5,756,507, 5,780,437 and 5,849,764.
Although dogs have been used to evaluate the efficacy of GnRH agonists and antagonists in vivo, the dog GnRH receptor has not been cloned and expressed in any cell lines. In this invention, we have cloned and sequenced the dog GnRH receptor and functionally expressed the receptor in a mammalian cell system. These tools facilitate the development of better GnRH agonists or antagonists for treating certain GnRH derived disorders.
This invention relates to the cloning and sequencing of the dog GnRH receptor. The DNA sequences disclosed herein may be engineered into expression systems designed for the production of the receptor and/or cell lines which express the receptor. Such cell lines may be used for screening and identifying compounds that function as GnRH agonists and antagonists.
Other aspects of this invention are nucleic acids which encode the receptor or a functional equivalent. These nucleic acids may be free from associated nucleic acids, or they may be isolated or purified. For most cloning purposes, cDNA is a preferred nucleic acid, but this invention specifically includes other forms of DNA as well as RNAs which encode the receptor or a functional equivalent.
A further aspect of this invention relates to vectors which comprise nucleic acids encoding the dog GnRH receptor or a functional equivalent. These vectors may be comprised of DNA or RNA; however, for most cloning purposes, DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes and other forms of episomal or integrated DNA that can encode the receptor. It is well within the skill of the ordinary artisan to determine an appropriate vector for a particular gene transfer or other use.
Other aspects of this invention are host cells which are transformed with a gene which encodes the dog GnRH receptor or a functional equivalent. The host cell may or may not naturally express the receptor on the cell membrane. Preferably, once transformed, the host cells are able to express the receptor or a functional equivalent on the cell membrane. Depending on the host cell, it may be desirable to adapt the DNA so that particular codons are used in order to optimize expression. Such adaptations are known in the art, and these nucleic acids are also included within the scope of this invention. Generally, mammalian cell lines, such as COS, HEK-293, CHO, HeLa, NS/O, CV-1, GC, GH3 or VERO cells are preferred host cells, but other cells and cell lines such as Xenopus oocytes or insect cells, may also be used.